The present invention concerns methods of transferring primordial germ cells to birds for the production of gametes therein. Such methods are useful in the conservation of endangered avian species, in reducing the time required to produce spermatozoa from slowly maturing species such as turkeys, decreasing the costs of maintaining breeder flocks, and altering the sex ratio of offspring flocks (e.g., to enhance the efficiency of production).
The ability to more easily produce gametes of particular avian species would be extremely useful to the avian veterinary and poultry production fields. For endangered species such as the whooping crane, it would be extremely useful to have a ready supply of male spermatozoa. For commercial birds such as turkeys, it would be desirable to more quickly and economically produce male spermatozoa. For meat- producing flocks, it is desirable to have ways to increase the ratio of male birds in the flock. Accordingly, there is a need for new ways to obtain avian spermatozoa.
Chimeras are composite organisms consisting of cells derived from more than one zygote. Experimental chimeras have been used to study cell to cell interaction and cell lineage analysis during development (A. McLaren, Mammalian Chimeras. Cambridge University Press, Cambridge (1976)). When chimeras are produced using material derived from very early embryos, organisms develop containing a full mixture of somatic tissues. If the starting material includes early germ cells or their precursors, the resulting individuals will produce gametes of both the donor and recipient genotypes. In addition, chimeras can be intraspecific, i.e. between two zygotes of the same species, or interspecific, i.e. between two different species.
Avian primordial germ cells (PGCs) like other vertebrate germ cells are extragonadal in origin and must undergo a complex journey to reach the gonad. The transfer of blastodermal cells and primordial germ cells has produced avian germline chimeras.
Reynaud (J. Embryol. Exp. Morphol. 21:485-507 (1969)), a pioneer in the production of avian germline chimeras, reported the production of turkey-chicken germline chimeras by the intravascular transfer of dissociated turkey germinal crescent cells into previously sterilized chick embryos (accomplished by exposure of the recipient germinal crescent to ultra-violet light). PGCs obtained by mechanical dissociation of the endoderm of the germinal crescent (stage 5) were injected into the blood vessels of chicken embryos (3-5 days of incubation). Prior to injection the recipient embryos were sterilized at stage 8-10 (HandH) with ultraviolet light; however, the sterilization was not complete. The turkey PGCs in the chick embryo were identified solely on the basis of their nucleoplasmic ratio. This method of identification was difficult and tenuous and could not be used for actively dividing turkey PGCs since the dividing germ cells gave an aberrant nucleoplasmic ratio. In a succeeding study, the transferred PGCs were allowed to undergo maturation in the host gonads and apparently could give rise to gametes but they were not suitable for fertilization (Wilhelm Roux Arch. Dev. Bio. 179:85-110 (1976)). The spermatozoa were incapable of fertilizing turkey eggs. They fertilized chick eggs but there was no normal development. Chicken spermatozoa were capable of activating the eggs obtained from female interspecific chimeras but they did not give rise to embryos. When the eggs were fertilized by turkey spermatozoa they developed into abnormal embryos that did not survive beyond stage 38 (HandH). Reynaud used morphology as the only distinguishing characteristic in an attempt to identify turkey germ cells from chicken germ cells. Morphology alone is not sufficient for identifying chimeras and must be substantiated with other markers. In addition, according to Aige-Gil and Simkiss (Brit. Poul. Sci. 32:427-438 (1991)), the presence of turkey gametes was not identified by test matings. Accordingly, there remains a need for new ways to accomplish the production and transfer of avian gametes.
A method for the production and collection of avian sperm comprises the steps of: providing primordial germ cells from a donor avian species; administering the primordial germ cells to a recipient avian species in ovo; incubating the recipient avian species to hatch; and then collecting sperm of the donor avian species from the recipient avian species. For example, the donor avian species may be a whooping crane, and the recipient avian species may be a sand hill crane. In another example, the donor avian species may be a turkey, and the recipient avian species may be a chicken.
In birds, unlike mammals, it is the male that is the homogametic sex (ZZ) and the female which is the heterogametic sex (Zw). Therefore in birds, it is the female that determines the gender of the offspring since she produces ova which carry either the Z or w chromosome. Thus, as noted below, by transferring male primordial germ cells to female embryonic hosts, the percentage of Z-bearing ova produced by that host is increased and the percentage of male offspring is increased. An increase in the percentage of male offspring from broiler flocks is economically desirable for the corresponding greater feed conversion ratio and more efficient meat production so obtained.
Accordingly, a second aspect of the present invention is a method of increasing the number of male birds hatched from a plurality of bird eggs, comprising the steps of: administering to a female bird in ovo male (ZZ) avian primordial germ cells; incubating the female bird to hatch; raising the female bird to sexual maturity; and then breeding the bird to produce a plurality of fertile bird eggs (with the ratio of male to female birds eggs produced from the bird being greater than that obtained in the absence of administering the male primordial germ cells to the bird in ovo.) Typically, the method further comprises the step of incubating the plurality of bird eggs to hatch (with the ratio of male to female birds produced from the plurality of eggs being greater than that produced in the absence of administering the male primordial germ cells to the female bird in ovo). The female bird may be of any suitable species, such as chicken or turkey, and the primordial germ cells being administered are preferably from the same species as the female bird to which they are administered.
The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.
xe2x80x9cBirdxe2x80x9d or xe2x80x9cavian speciesxe2x80x9d as used herein refers to any avian species, including but not limited to chicken, turkey, duck, geese, quail, pheasant, and ostrich. Any of numerous other species can be employed to carry out the present invention, particularly when it is used for the conservation of endangered species such as the whooping crane (where the recipient species would be the sand hill crane). xe2x80x9cEggxe2x80x9d as used herein refers to avian eggs that contain live embryonic birds. xe2x80x9cPrimordial germ cellxe2x80x9d or xe2x80x9cPGCxe2x80x9d as used herein refers to the most differentiated diploid cell line in the embryo that will ultimately develop into haploid gametes (either sperm or ova).
xe2x80x9cSSEA-1 antibodyxe2x80x9d refers to an antibody, preferably a monoclonal antibody, that specifically binds to the stage specific embryonic antigen-1 (SSEA-1) (M. Buehr Exp. Cell Res. 232, 194-207 (1997)). SSEA-1 is a carbohydrate epitope determined by galactose xcex21xe2x86x924 fucose xcex11xe2x86x923 N acetylglucosamine linkage (H. Gooi et al., Nature 292, 156-158 (1981)). A monoclonal antibody to SSEA-1 was developed by the fusion of mouse myeloma cells with spleen cells from a mouse that had been immunized with F9 teratocarcinoma cells (D. Solter and B. Knowles, Proc. Natl. Acad. Sci. USA 75, 5565-5569 (1978)). SSEA-1 antibody is known as an avian immunohistochemical germ cell marker (L. Karagenc et al., Dev. Genet. 19, 290-301 (1996)). Particularly preferred is clone MC 480, which may be obtained from the Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, Iowa, USA.
Primordial germ cells may be provided and formulated for carrying out the present invention by any suitable technique, and stored, frozen, cultured or the like prior to use as desired. For example, the primordial germ cells may be collected from donor embryos at an appropriate embryonic stage (see, e.g., (V. Hamburger and H.L. Hamilton, A Series of Normal Stages in the Development of the Chick, Journal of Morphology, 88, 49-92 (1951) (These stages referred to as HandH stages herein) stage 4, or the germinal crescent stage, through stage 30, with cells being collected from blood or gonad in the later stages). The primordial germ cells are, in general, twice the size of somatic cells and easily distinguished and separated therefrom on the basis of size. Male (or homogametic) primordial germ cells (ZZ) can be distinguished from heterogametic primordial germ cells (Zw) by any suitable technique, such as collecting germ cells from a particular donor and typing other cells from that donor, the collected cells being of the same chromosome type as the typed cells. Cell may be formulated for administration to animals by dissociating the cells (e.g., by mechanical dissociation) and intimately admixing the cells with a pharmaceutically acceptable carrier (e.g., phosphate buffered saline solution). The primordial germ cells are preferably gonadal primordial germ cells or blood primordial germ cells (xe2x80x9cgonadxe2x80x9d or xe2x80x9cbloodxe2x80x9d referring to their tissue of origin in the original embryonic donor), and are most preferably gonadal primordial germ cells. The primordial germ cells administered may be heterogametic (Zw) or homogametic (ZZ) depending upon the particular object of the administration. PGCs are preferably administered in physiologically acceptable carrier, preferably at a pH of from about 6 to about 8 or 8.5, in a suitable amount to achieve the desired effect (e.g., 100 to 1000 PGCs per embryo). The PGCs may be administered free of other ingredients or cells, or other cells and ingredients may be administered along with the PGCs.
Administration of the primordial germ cells to the recipient animal in ovo may be carried out at any suitable time at which the PGCs can still migrate to the developing gonads. In general, it is preferred that administration be carried out from stage 13 or 14 through stage 18 (HandH) of embryonic development, and most preferably stage 15. For chickens, the time of administration is thus during days 1, 2, 3 or 4 of embryonic development, most preferably day 2 to day 2.5. Administration is typically by injection into any suitable target site, such as the region defined by the amnion (including the embryo), the yolk sac, etc. Injection into the embryo itself (including the embryo body wall) is preferred, and intravascular or intracoelomic injection into the embryo are particularly preferred. The methods of the present invention may be carried out with or without prior sterilization of the recipient bird in ovo. (by xe2x80x9csterilizationxe2x80x9d is meant render substantially incapable of producing gametes). In a preferred embodiment of the invention, the primordial germ cells are conveniently administered to a recipient subject in ovo that has not been previously sterilized. When donor gametes are collected from such a recipient, they may be collected as a mixture with gametes of the donor, and may be used as such a mixture or the mixture processed to enrich the proportion of donor gametes therein.
Administration of PGCs may be carried out by administering PGCs per se, or by administering precursors cells that develop into PGCs in the subject (particularly where the invention is employed to alter the sex ratio of offspring). For example, administration may be carried out by injecting the bird with blastodermal cells, where the blastodermal cells differentiate into primordial germ cells in vivo in the bird.
When used for the production and collection of avian gametes (sperm, ova), the primordial germ cells are administered in ovo to a recipient species that is different from the donor species from which the PGCs were obtained. The recipient is then incubated to hatch and raised to sexual maturity, and sperm cells or ova of the donor species collected from the recipient animal, all in accordance with standard techniques. For example, in the case of an endangered species, the donor avian species may be a whooping crane, and the recipient avian species may be a sand hill crane. In another example concerning commercial poultry production, the donor avian species may be a turkey, and the recipient avian species may be a chicken.
When used for increasing the number or ratio of male birds hatched from a group of eggs, the present invention involves administering to a female bird in ovo male avian primordial germ cells. The gender of the bird in ovo may be predetermined or determined after hatch. The bird is then incubated to hatch, the gender of the bird determined if necessary, raised to sexual maturity, and bred by crossing the bird with a suitable male breeder stock in accordance with known techniques. A plurality of fertile eggs laid by that bird are then collected, and typically incubated to hatch and the resulting birds grown for at least two to three weeks. The ratio of male to female bird eggs (or birds) produced from the female bird is greater than that obtained in the absence of administering the male primordial germ cells to that bird in ovo. Such methods are typically used on species of bird that are raised for meat production, such as chickens, turkeys, ducks, etc.
The in ovo administration of the primordial germ cells may be carried out by any suitable technique, either manually or in an automated manner. Injection is preferred. The mechanism of in ovo administration is not critical, but it is preferred that the method not unduly damage the tissues and organs of the embryo or the extraembryonic membranes surrounding it so that the treatment will not unduly decrease hatch rate. A hypodermic syringe fitted with a needle of about 18 to 26 gauge is suitable for the purpose. Depending on the precise stage of development and position of the embryo, a one-inch needle will terminate either in the fluid above the chick or in the chick itself. A pilot hole may be punched or drilled through the shell prior to insertion of the needle to prevent damaging or dulling of the needle. If desired, the egg can be sealed with a substantially bacteria-impermeable sealing material such as wax or the like to prevent subsequent entry of undesirable bacteria. It is envisioned that a high speed injection system for avian embryos will be particularly suitable for practicing the present invention. Numerous such devices are available, exemplary being the EMBREX INOVOJECT(trademark) system (described in U.S. Pat. Nos. 4,681,063 and 4,903,625 to Hebrank), and U.S. Pat. Nos. 4,040,388; 4,469,047, and 4,593,646 to Miller. The disclosure of all United States patent references cited herein are be incorporated herein by reference in their entirety. All such devices, as adapted for practicing the present invention, comprise an injector containing the a formulation of the primordial germ cells as described herein, with the injector positioned to inject an egg carried by the apparatus in the appropriate location within the egg as discussed above. In addition, a sealing apparatus operatively with the injection apparatus may be provided for sealing the hole in the egg after injection thereof.